DAT is a neurotransmitter transporter that clears the synapse of dopamine (DA) following neural depolarization and synaptic release. Protein kinase C activation stimulates DAT phosphorylation and internalization and reduces DA uptake. The purpose of this project is to examine and characterize DAT phosphorylation and functional regulation in response to amphetamines, cocaine and other psychostimulants. Specific Aim One characterizes DAT phosphorylation in response to DAT substrates or uptake blockers by metabolic labeling of DATs expressed in rat striatum or cultured cells Specific Aim Two examines PMA/Oainduced DAT phosphorylation in response to DAT substrates or uptake blockers. Specific Aim Three will examine DAT function by [3H]DA uptake and cell surface expression of DAT by biotinylation, in response to treatments from Specific Aims One and Two that modulate DAT phosphorylation. The purpose of this research proposal is to advance the understanding of DAT phosphorylation and functional regulation with respect to abused and neurotoxic drugs.